Separation of DNA restriction fragments by FPLC ion-exchange chromatography on Mono Q and Mono P columns was investigated. The columns were found to be particularly suitable for the separation of fragments up to 500–600 bp long. Larger fragments can also be separated although less effectively. We found the following practical working ranges for the parameters investigated: pH, 4 to 11; flow rate, 0.05 to 0.6 ml/min corresponding to separation times between 2 and 20 h. (better resolution is achieved at lower flow rates); gradient slope; between 0.5 m m eluting salt/ml buffer and over 5 m m/ml (better resolution is achieved at lower gradient slopes; eluting ionic strength was found to be independent of gradient slope); gradient composition, chloride salts of smaller monovalent cations eluted the DNA at lower ionic strengths but separations obtained were similar; additives, substances such as urea, formamide, and EDTA can be added without chromatographic effects; sample amount: amounts from 2.5 to 200 μg were applied, corresponding to single peak content of from 42 ng to 74μg DNA. Yields were generally over 90% and the chromatographed DNA was fully accessible to restriction enzyme cleavage. Separations occurred predominantly according to DNA size, but AT-rich fragments were retarded in a predictable way.